Download Crystal structure of ATP sulfurylase from Saccharomyces cerevisiae

The EMBO Journal Vol. 20 No. 3 pp. 316±329, 2001
Crystal structure of ATP sulfurylase from
Saccharomyces cerevisiae, a key enzyme in sulfate
activation
Tobias C.Ullrich1, Michael Blaesse and
Robert Huber
Max-Planck-Institut fuÈr Biochemie, Abteilung Strukturforschung,
Am Klopferspitz 18a, D-82152 Martinsried, Germany
1
Corresponding author
e-mail: [email protected]
ATP sulfurylases (ATPSs) are ubiquitous enzymes
that catalyse the primary step of intracellular sulfate
activation: the reaction of inorganic sulfate with ATP
to form adenosine-5¢-phosphosulfate (APS) and pyrophosphate (PPi). With the crystal structure of ATPS
from the yeast Saccharomyces cerevisiae, we have
solved the ®rst structure of a member of the ATP sulfurylase family. We have analysed the crystal strucÊ resolution using
ture of the native enzyme at 1.95 A
multiple isomorphous replacement (MIR) and, subsequently, the ternary enzyme product complex with
APS and PPi bound to the active site. The enzyme consists of six identical subunits arranged in two stacked
rings in a D3 symmetric assembly. Nucleotide binding
causes signi®cant conformational changes, which lead
to a rigid body structural displacement of domains III
and IV of the ATPS monomer. Despite having similar
folds and active site design, examination of the active
site of ATPS and comparison with known structures
of related nucleotidylyl transferases reveal a novel
ATP binding mode that is peculiar to ATP sulfurylases.
Keywords: adenosine-5¢-phosphosulfate/ATP sulfurylase/
crystal structure/sulfate activation/sulfur amino acid
biosynthesis
Introduction
ATP sulfurylase (ATPS) (adenylsulfuryltransferase/
ATP:sulfate adenylyltransferase; E.C. 2.7.7.4) is the ®rst
catalytic enzyme in a cascade of proteins that performs the
activation of inorganic sulfate (SO42±) and its incorporation into organic compounds. ATPS catalyses adenylylsulfate synthesis, the ®rst intracellular reaction in sulfate
assimilation. Sulfate is activated with ATP to give
adenosine-5¢-phosphosulfate (APS) and inorganic pyrophosphate (PPi) according to the scheme:
MgATP + SO42± ® MgPPi + APS
This reaction yields a molecule with a high energy
mixed phosphoric±sulfuric acid anhydride bond, which is
used for further sulfate activation and reduction in the cell.
Thus, ATPS plays an important role in sulfate activation
as a component of the sulfur amino acid (methionine/
cysteine) biosynthesis pathway. This constitutes part of a
global regulatory circuit, which controls the expression of
316
enzymes that function both in the acquisition of sulfur
from the environment and its assimilation (Marzluf, 1997).
In sulfate-reducing bacteria, ATPS plays a key role not
only in amino acid metabolism, but also in anaerobic
dissimulation, where ATP production is linked to APS
reduction to AMP and sul®te, which is further reduced to
hydrogen sul®de (Gavel et al., 1998; Sperling et al., 1998).
In bacteria such as Escherichia coli, ATPS is a
tetramer of heterodimers. Each heterodimer of K-12 is
composed of two subunits, CysD and CysN (Leyh
et al., 1988). The GTPase subunit CysN activates the
catalytic subunit CysD by allosteric interactions (Wei
et al., 2000). ATPS from sulfate-reducing bacteria and
eukaryotic organisms such as yeast (Karamohamed
et al., 1999), ®lamentous fungi (Foster et al., 1994)
and plants (Leustek et al., 1994; Logan et al., 1996)
have been described as monomers or homo-oligomeric
complexes, which do not require GTP for activation.
However, in higher eukaryotes, like the marine worm
(Rosenthal and Leustek, 1995) and mammalian species
(Li et al., 1995; Venkatachalam et al., 1998), gene
fusion has occurred; ATPS and the APS kinase, which
performs the ®nal step of the synthesis of the highenergy sulfate donor PAPS (3¢-phosphodenosine5¢-phosphosulfate) by 3¢-phosphorylation of APS, are
arranged as subdomains on a single polypeptide chain.
Together they compose the bifunctional PAPS synthetase, which in mammalian cells, besides its role in
amino acid metabolism, is also important for the
sulfation of proteins, carbohydrates, lipids, drugs and
xenobiotics. The fusion of ATPS and APS kinase into
one molecule represents an evolutionary trend towards
more functional ef®ciency in sulfate activation and
amino acid metabolism in higher organisms, providing
a mechanism for channelling the unstable APS intermediate into the adjacent kinase reaction.
ATPS of the yeast S.cerevisiae is encoded by the MET3
gene on chromosome X (Cherest et al., 1987). In contrast
to the related nucleotide-binding kinases, which catalyse
the removal of the g-phosphate of an NTP, it can be
classi®ed into the superfamily of a/b phosphodiesterases
(pyrophosphorylases, ATP PPases) due to its catalytic
activity described above. Similarities in its sequence, like
the typical HxxH motif, also bring the ATPS into close
proximity with the superfamily of nucleotidylyl transferases, such as the prototypical Bacillus subtilis cytidylyltransferase TagD/GCT (CTP:glycerol-3-phosphate
cytidylyltransferase) (Park et al., 1997; Weber et al.,
1999) or the related class I aminoacyl-tRNA synthetases
(for review see Delarue and Moras, 1993), of which
several structures are known. All these functionally
characterized enzymes cleave the a-b-phosphate bond of
an NTP and transfer the NMP to the second substrate.
ã European Molecular Biology Organization
Crystal structure of ATP sulfurylase
Fig. 1. Sequence alignment of ATP sulfurylase from S.cerevisiae with ATPS of P.chrysogenum, Z.mays and A.fulgidus, and the ATP sulfurylase
subdomains of the two isoforms of mouse and human PAPS synthetase. Sequence identities between yeast and fungi ATPS are coloured in blue, and
those between yeast ATPS and all others are in red. Less conserved regions are marked in yellow. The highly conserved RNP and GRD motifs are
highlighted in green boxes, with the histidines of the HxxH motif drawn in additional rectangles. The secondary structure symbols are set according to
the presented ATPS structure.
Figure 1 shows the alignment of yeast ATPS
with homologues from Penicillium chrysogenum,
Archaeoglobus fulgidus and the C-terminal ATPS
domains of maize (Zea mays), mouse and human
PAPS synthetase isoforms, all of which show strong
homologies. In particular, the correspondence of 66%
of the amino acids from yeast ATPS to ATPS from
the ®lamentous fungi P.chrysogenum, which is mainly
evident for the ®rst 400 amino acids, is notable. The
C-terminal region of yeast ATPS, however, is very
distinct from the fungal ATPS, which shows strong
sequence homologies to the APS kinases, indicating
that they may have evolved from APS kinase (MacRae
and Segel, 1997). Although the higher eukaryotic
PAPS synthetases from maize and mammals show
sequence similarities of up to 70% with yeast ATPS,
they do not have the C-terminal 100-amino-acid
extensions of yeast and fungal ATPS.
The identities of yeast ATPS to the PAPS
synthetases of both mouse and human isoforms are
28% in all four cases, and still 20% to the less-related
archaebacterium A.fulgidus. In spite of this, in all the
presented sequences prominent motifs can be highlighted: the HxG/AH motif and the subsequent Leu207
of the ATPS sequence are present together with the
preceding residues 190VxAFQxRNP, which are also
317
T.C.Ullrich, M.Blaesse and R.Huber
Table I. Concentration, soaking time and data collection statistics
Native
TaBr
Hg1
URA
APS
PPi
Reactant
Ta12Br14
Thiomersal
UO2Ac2
MgATPgs
MgATPgs/KClO4
Concentration (mM)
Soaking time (h)
Asymmetric monomers
Ê)
Wavelength (A
Ê)
Limiting resolution (A
Unique re¯ections
Ê)
Last shell (A
Completeness (%)
Mean redundancy
Mean I/sI
Rmerge
Phasing power
Sites
5
12
1
1.2539
2.2
35 064
2.2±2.37
96.3 (96.3)
8.5 (8.3)
7.4 (1.5)
6.8 (40.2)
1.04
1
10
12
1
1.54
2.5
26 716
2.5±2.64
98.8 (98.8)
3.8 (3.7)
6.5 (1.1)
9.6 (64.6)
0.32
1
5
3
1
1.54
2.7
21 297
2.7±2.85
99.6 (99.9)
5.3 (5.3)
5.6 (1.8)
12.2 (40.5)
0.89
1
5
3
2
1.54
2.6
45 486
2.6±2.76
99.5 (97.3)
6.0 (5.6)
6.4 (1.8)
11.0 (41.3)
±
±
5/10
3
2
1.54
2.8
35 298
2.8±2.98
95.5 (97.1)
4.1 (3.8)
5.8 (1.6)
12.8 (42.8)
±
±
1
1.04
1.95
55 315
1.95±2.07
98.3 (98.6)
3.8 (3.7)
9.3 (2.7)
5.1 (28.0)
±
±
Rsym = S|I(h)i ± <I(h)>|/S<I(h)>
Table II. Re®nement statistics
Space group
Cell constants
a
b
c
a
b
g
Ê)
Resolution range (A
Re¯ections in working set
Re¯ections in test set
Rcryst (%)
Rfree (%)
Protein atoms (non-H)
Nucleotide atoms
Heterogen atoms (non-H)
Solvent molecules
Ê 2)
Average B-factor (A
R.m.s.ds
Ê)
bond lengths (A
bond angles
Ê 2)
DB (through bonds) (A
Rcryst
free
Native
APS
complex
APS±PPi
complex
R32
R32
R32
187.1
187.1
115.9
90
90
120
20.0±1.95
55 315
2835
19.7
23.2
4077
±
73
566
35.4
185.9
185.9
223.7
90
90
120
25.0±2.6
45 468
2337
17.6
22.7
8154
54
120
763
35.1
186.0
186.0
223.7
90
90
120
25.0±2.8
35 298
1801
19.3
24.4
8154
63
120
600
35.9
0.0104
1.49
1.83
0.0097
1.56
1.72
0.0068
1.32
1.42
= S|Fobs ± Fcalc|/S|Fobs|
strictly conserved in all the aligned sequences. Also,
the module 288VGRDHAG, which from now on will be
referred to as the GRD-loop, is highly conserved. It is
known to play a crucial role in substrate binding,
analogous to the PP-loop motifs of other nucleotidylyl
transferases. Many site-directed mutagenesis studies
with ATPS and PAPS synthetases of several organisms
(Deyrup et al., 1999; Venkatachalam et al., 1999), as
well as with the related cytidylyltransferases (Park
et al., 1997), demonstrate that alanine mutants of the
above speci®ed residues produce either reduced activity
or inactive enzymes. This strongly indicates that
nucleotide binding as well as sulfurylase activity
involves residues of these motifs. Additionally, a
fourth highly conserved motif can be found: the
356ISGTxxR363
module is widespread among the
318
aligned eukaryotic species, but has not yet been
investigated for its detailed function.
To support and extend the extensive biological and
biochemical studies, structural investigations were initiated to elucidate the architecture and functional features of
ATPS, with the aim of identifying the active site and the
catalytic residues involved in sulfate activation. In this
work we present the puri®cation, crystallization and
determination of the three-dimensional (3D) structure of
ATPS from S.cerevisiae.
Results and discussion
We have developed a procedure to purify 5±10 mg of
ATPS from 500 g of yeast cells to electrophoretic
homogeneity. The molecular weight of the puri®ed
protein was determined to be ~58 kDa for the
protomer. Further analysis has shown that ATPS is a
homo-oligomer with a molecular weight of
350±400 kDa, suggesting a homohexameric assembly.
Crystallization experiments with puri®ed ATPS yielded
trigonal crystals that belong to the spacegroup R32.
The protein crystallizes reproducibly within 2 days
from solutions containing Cd2+ as an additive, with
average dimensions of 0.5 3 0.5 3 0.3 mm3, and with
Ê (native enzyme) and
crystals diffracting up to 1.95 A
Ê (product complex), respectively. For the native
2.6 A
crystals, the asymmetric unit contains one subunit of
ATPS, resulting in a Matthew's coef®cient of 3.38
A3/Da (Matthews, 1968) and a solvent content of 64%.
As shown in Table II, the crystals of the enzyme±
product complexes contain two independent subunits in
the asymmetric unit in the same trigonal R32 spacegroup as the native crystals, indicating a signi®cant
change in the crystallographic packing.
The 3D structure of ATPS was determined by
multiple isomorphous replacement (MIR) using tantalum bromide, mercury (thiomersal) and uranyl acetate
as heavy-atom derivatives (Table I). An initial polyÊ
alanine model was built in the MIR density at 2.7 A
resolution. By phase combination of the model and the
Ê , the model
MIR phases, and their expansion to 1.95 A
could be completed using the amino acid sequence of
Crystal structure of ATP sulfurylase
Fig. 2. (A) Stereo ribbon plot of the ATPS homohexamer, top view, and (B) side view. (C) The side view rotated 30° around the 3-fold axis vertical
in the paper plane, with a black arrow marking the non-equivalence of the rings in the complex structure. (D) Stereoview of the ATPS protomer in
ribbon presentation (top view). The four individual domains are labelled in grey, red, yellow and cyan (from domain I to IV). Domain II is shown
with an APS molecule (yellow, ball-and-stick) in the active site.
S.cerevisiae ATPS from the strain S288C/FY1679
(M.de Haan et al., 1995). The resulting native structure
contains all 510 residues, without the initial methionine, 11 metal ions, two sulfates, 11 acetate ions and
one Tris molecule as ligands. The ®nal R-factor for
the native model is 19.6% (Rfree 23.1%). The complex with APS was subsequently solved using molÊ
ecular replacement techniques and re®ned at 2.6 A
resolution with an R-factor of 17.6% (Rfree 22.7%).
Analysis of the stereochemistry with PROCHECK
(Laskowski et al., 1993) revealed that 91.2% of the
dihedral f/y angles lie in the most favoured regions of
the Ramachandran plot, 8.4% in the additional allowed
regions and 0.5% in the generously allowed regions.
Further details are given in Materials and methods and
in Table II.
Quaternary structure
The quaternary arrangement of ATPS is generated by the
D3 symmetry of the R32 spacegroup, resulting in a
homohexameric assembly, which had been suggested for
the homologue of ATPS from P.chrysogenum. Its molecular weight of ~350 kDa is in accordance with earlier
solution studies. In the D3 symmetric structure, two
319
T.C.Ullrich, M.Blaesse and R.Huber
trimeric rings assemble in a staggered con®guration with a
twist of 60°. ATPS exhibits overall dimensions of
Ê 3. Side and top views of the
150.6 3 133.3 3 81.6 A
particle are shown in Figure 2A±C.
Monomer structure
As illustrated in Figure 2D, the U-shaped protomer
consists of four easily distinguishable domains and is
characterized by a high ratio of regular secondary structure
elements.
Domain I is built by the N-terminal part of the peptide
chain (residues 2±167) and comprises as a main motif a
b-barrel, which is formed by ®ve antiparallel strands in a
1-3-4-5-2 topology. It is topologically similar to the
pyruvate kinase b-barrel domain (Figure 3A), but in
contrast to the pyruvate kinase b-barrel, which is
characterized by an all-b conformation, ATPS shows a
more complex folding topology with several helical
insertions such as an a±turn±a module (helices H6 and
H7) and an additional short b-hairpin motif formed by S3
and S4 after helix H4, making it more similar to an a/b
protein. The inner cage of the b-barrel of ATPS is ®lled
with hydrophobic residues to build a large hydrophobic
network. Domain I and the adjacent domain II face each
other with a large planar surface of mostly polar or charged
residues. These interactions are predominantly mediated
by hydrogen bonds and salt bridges. Contacts between the
residues of helix H3 and the coiled region preceding H11
of the interdomain show a more hydrophobic character.
Domain II (residues 168±327) comprises the active site
and the substrate binding pocket. It shows a typical righthanded a/b-fold in three layers (Rossmann-like fold;
Rossmann et al., 1974), which is shared by members of the
superfamilies of adenine nucleotide a hydrolases and of
nucleotidylyl transferases, e.g. GMP synthetase or the
class I aminoacyl-tRNA synthetases and cytidylyltransferases. Five twisted parallel b-strands (S9±S13) with a
3-2-1-4-5 topology are ¯anked by ®ve a-helices to form
the typical a/b/a supersecondary structure. Figure 3B
shows a ribbon plot superimposition of ATPS domain II
with the structure of the CTP:glycerol-3¢-phosphate
cytidylyltransferase from B.subtilis (Weber et al., 1999),
a typical member of the nucleotidylyl transferase a/b
phosphodiesterase superfamily. Using TOP (Lu, 2000) for
structure similarity search, CGT was found to ®t best with
Ê (matcha root mean square deviation (r.m.s.d.) of 1.57 A
ing residues). But, in addition to these common elements,
ATPS exhibits a large loop motif of b-turns between S12
and the helix H12 (residues 288±306). Domain II is packed
against domain III mainly by charged and hydrophilic
residues of H9 and H10, which are hydrogen bonded to
extended parts of the whole domain III.
Domain III consists of the residues 328±393 and is a
short domain of 65 amino acids. It links the C-terminal
domain IV with domains I and II, and its fold shows no
similarities to known protein families (Figure 2D).
Residues 327±331 participate in the formation of the
upper part of the ATPS substrate binding pocket. The
highly conserved 327PFR motif between strands S13 and
S14 displays some ¯exibility when the enzyme binds ATP,
causing a conformational change of only a few amino
acids, which is associated with the rigid body motion of
the entire domains III and IV. Flexibility is also manifested
320
in the electron density of the coiled region after the
antiparallel strands S14 and S15 (residues 347±352),
which shows weakly de®ned spheres in the native
Ê 2, e.g.
structure, with temperature factor values of ~75 A
for Lys350, but is well de®ned in the product complex.
The C-terminal domain IV again displays an a/b-fold
(residues 394±511) with ®ve twisted parallel b-strands
(S16±S20), but with a 2-3-1-4-5 topology. This structural
classi®cation reveals its close relationship to the superfamily of P-loop-containing nucleotide triphosphate
hydrolases and the family of nucleotide kinases, indicating
a common evolutionary origin with APS kinase. Typical
members of this family are yeast guanylate and uridylate
kinase. Despite their common fold topology, the superimposition of domain IV with the structure of uridylate
kinase from S.cerevisiae (Figure 3C) shows that the kinase
chain possesses additional extensions, such as a multiple
a-helix motif between the strands S19 and S20. This large
region participates in ATP binding and the formation of
the kinase reaction centre. The lack of this module, which
is also present but disordered in the APS kinase structure
(MacRae et al., 2000), provides striking evidence for the
absence of PAPS binding capability and a putative kinase
activity of ATPS. The C-terminus at Phe511 binds to
Pro250 by hydrophobic side-chain interactions, and with
its carboxylate group to Arg248-Nz of the neighbouring
protomer in the same ring.
Within the homohexamer, interactions of the protomers
are predominantly mediated through numerous hydrogen
bonds and salt bridges, including a broad network of
solvent molecules. In addition, we found a number of
metal ions contributing to the formation of the monomer±
monomer interfaces. They are all characterized by clearly
de®ned high electron density spheres, short distances to
the coordinating ligands and high coordination numbers.
Because of the presence of 25 mM cadmium in the
crystallization medium, these metal ions were interpreted
as cadmiums. Cadmium Cd515, for example, which
connects one protomer of the upper ring with a subunit
of the lower ring of the same particle, is coordinated to
Glu182-Oe and to Gly171-O from the upper subunit,
which makes further hydrogen bonds to Lys174-Ne. Both
residues are linked to the acetate Acy533 from the
protomer of the lower ring. Metal ions also connect
interfaces to neighbouring subunits of the same ring, such
as Cd516, which is 6-fold coordinated by the carboxylate
oxygens of two acetates, Acy527 and Acy534, to the
Asp189 and ®nally to His494-Ne, which is contributed
from the symmetry-related subunit. Some of these metal
sites may play a role in physiological conditions, whereby
calciums replace the cadmium ions. Two more metal sites
are located on the surface of the molecule. Due to its
coordination to the carboxylate oxygens of the highly
conserved Asp168, as well as to a sulfate ion, and to
Ê,
His235-Ne and His236-Ne at distances of 2.42 and 2.37 A
respectively, the ®rst metal Cd512 was interpreted as a
cadmium that may be zinc or cobalt under physiological
conditions, in accordance with previously described zinc
binding sites in ATPS of sulfur-reducing bacteria (Gavel
et al., 1998). Being located far away from the active site
cleft, it does not seem to ful®l any role in the catalytic
mechanism or in substrate binding, but it may contribute to
the structural integrity of the monomer and stabilize the
Crystal structure of ATP sulfurylase
Fig. 3. Stereoviews of ATPS domains I, II and IV (helices, red; strands, yellow; coiled regions or turns, grey) in ribbon presentation showing a
structural comparison of the typical fold motifs of the single domains with the best-®tting members of the related superfamilies (cyan). (A) Domain I
superimposed with the pyruvate kinase b-barrel domain. (B) Superimposition of domain II with GCT, a typical member of the superfamily of
nucleotidylyl transferases. Both polypeptides show the typical a/b-fold of the nucleotidylyl transferases. (C) Overlay of the structure of domain IV
with yeast uridylate kinase, complexed with ADP. Both polypeptides show the typical nucleotide and nucleoside kinase fold. All ATPS secondary
structure elements are labelled.
complex structure. Additionally, the Cys43-S and the
Pro39-O serve, along with four water molecules, as
ligands for cadmium Cd513 to form a slightly distorted
octahedral complex. Cadmium Cd514, however, mediates
interactions between the hexamers in the crystallographic
packing and is coordinated to the carboxylate oxygens of
Glu22, to Leu18-N, as well as to the acetate Acy526 and to
one water molecule. From the other subunit, His319-Ne
321
T.C.Ullrich, M.Blaesse and R.Huber
compose the pocket for the nucleosyl moiety of the
ATP/APS.
The crystal structure of ATPS was initially solved in the
absence of nucleotide; however, the crystallization
required 25 mM CdSO4 as an essential additive. Due to
the presence of sulfate in the crystallization buffer, a
sulfate, which is one of the substrates of ATPS, along with
several water molecules were found in the active site
Ê 2, the
(Figures 4 and 5A). With an average B-factor of 37 A
sulfate ion exhibits well-de®ned density and is coordinated
by three amino acids and several solvent molecules: O1 is
hydrogen bonded only to WAT779, whereas O2 is linked
to Ala293-N and a water molecule. The Arg197-Nz as
well as one water molecule interacts with O3, whereas the
oxygen O4 shows a hydrogen bond to Gln195-Ne. Thus,
the negative charge of the sulfate appears well balanced
and we assume that the sulfate occupies the regular
substrate binding site.
The adenylylsulfate complex
Fig. 4. Electrostatic surface potential plot of the ATPS protomer
(positive, blue; negative, red) showing the entrance to the active site of
the native molecule with a sulfate located in the substrate binding
pocket. The funnel leading into the bowl-like cleft is mainly positively
charged.
serves as a ligand completing the octahedral coordination
sphere. Other metals found in the density are supposed to
be smaller cations like sodium, calcium or magnesium,
compensating negative charges on the protein surface.
Nevertheless, they are inconsistent throughout the examined structures and, therefore, not described in detail.
The active site and nucleotide binding
As shown in Figure 4, the active site lies as a deep, bowllike cleft in the centre of the ATPS protomer. It consists
mainly of the domain II secondary structure elements H9,
S9, S10 and S12, as well as the H9 preceding RNP-loop
from Phe194 to His201 and the GRD-loop, which are both
highly conserved. Moreover, the domain III residues
Pro327, Phe328 and Arg329, together with Met330 and
Val331, contribute to the formation of the cleft, which is
open to the top of the molecule but faces the inner
compartment of the U-shaped protomer (Figure 5A). It
Ê in length and
reaches maximum dimensions of 19.5 A
Ê in width, and has, unlike some other nucleotidylyl
17.5 A
transferases, no contact with symmetry-related subunits.
The funnel directing into the cleft is lined with a bundle of
positively charged residues to build an anion hole for the
incoming nucleotide. While the bottom and side walls of
the cleft are mainly formed by either hydrophilic or
positively charged residues, which are supposed to play a
crucial role in phosphate/sulfate binding or even in the
catalytic mechanism, the back of the pocket is hydrophobic in character, consisting of the highly conserved
residues Phe194, Leu207, Ile287, Val288, Gly289,
His292, Phe328 and Met330. These residues interact
together with the uncharged residues Val191±Ala193 as a
network in the inner compartment of the domain to
322
In order to obtain further information about substrate
binding and the mechanism of sulfate activation, we
attempted to determine the structure of ATPS complexed
with its second substrate ATP. We performed soaking
experiments with the apo-crystals and the ATP analogues,
MgATPgs and MgATPas, which are both unhydrolysable
or at least very slowly hydrolysed to AMP or ADP,
respectively. Surprisingly, we obtained identical results
from both trials: the 2Fo ± Fc Fourier map, calculated
using the phases from the re®ned model of ATPS, shows
strong positive density in the active site cleft, which can
clearly be ®tted with an adenosyldinucleotide molecule
lacking the g-phosphate (Figure 5B). Two possible
explanations for this observation could be the spontaneous
hydrolysis of both ATP analogues in solution, or disorder
of the g-phosphate in the crystal packing. But, as shown in
Figure 5B and in the schematic drawing (Figure 6), the
nucleotide is well coordinated by the surrounding enzyme
residues so that the pocket does not allow free rotation of
the g-phosphate. Also, the hydrolysis of the relatively
stable ATP analogues in solution or by enzymatic catalysis
does not seem to be a very plausible explanation for this
observation, especially since ATPS has no phosphorylase
activity. It is more likely that the enzymatic reaction has
occurred in the crystal and that the density represents
Ê 2 for the
the product APS. An average B-factor of 31.3 A
whole APS molecule compared with a main chain B-factor
Ê 2 indicates strong binding. The APS adopts an
of 32.1 A
open L-shaped conformation, with the adenosyl moiety
protruding deeply into the space between helix H9 and the
b-strands S9 and S12. The sulfate group, oriented to the
right side of the a-phosphate, is coordinated to Gln195Ne, Arg197-Nz and Ala293-N in the same pocket, as
described above for the native state. The a-phosphate is
®xed between the residues Thr196, Arg197 and Asn198.
Whilst Arg197 coordinates to the a-phosphate oxygen via
its peptide nitrogen, Thr196 and Asn198 form hydrogen
bonds with their side-chain hydroxyl groups. The conserved Gly289 of the GRD-loop allows close approach of
the main chain to the APS ribose moiety, with the contacts
to the ribose unit being formed by backbone interactions
of Gly289-N and His292-O to the 3¢-oxygen and of
Arg290-O to the 2¢-oxygen, respectively. Additionally, the
Crystal structure of ATP sulfurylase
Ê (A), the binary enzyme product complex with APS at 2.6 A
Ê (B), and
Fig. 5. Active site residues of the apo-protomer liganded with sulfate at 1.95 A
Ê (C). All atoms are shown with the ®nal 2Fo ± Fc electron density contoured
the ternary product complex with an additional pyrophosphate at 2.8 A
at 1.0s.
adenyl moiety is associated with Val331-N and Met330-S
by hydrogen bonds and shows van der Waals contacts to
numerous other residues. Further details of these interactions are depicted schematically in Figure 6.
Nucleotide binding causes a signi®cant rigid body
displacement within the ATPS hexameric assembly. This
rearrangement of protomer subdomains and, accordingly,
of the entire enzyme complex seems to be caused by one
single amino acid of the 327PFR motif (Figure 7). In the
absence of a nucleotide, the phenyl ring of Phe328, which
marks a hinge point for the domain movements, is stuck
deeply in the hydrophobic adenyl binding site. When ATP
323
T.C.Ullrich, M.Blaesse and R.Huber
Fig. 6. Schematic drawing of the coordination of APS in the binding pocket, its hydrogen bonding and hydrophobic interactions with solvent
Ê.
molecules and the residues of ATPS. Bond distances are given in A
binds, the phenyl ring is displaced from its position in the
unliganded molecule and is pushed out to the top of the
pocket. The ¯ipping of the Phe328 by 90° induces a
reorientation of the backbone and side chains of the
neighbouring Pro327 and Arg329, with residue Met330
Ê and creating, together with the side
being shifted by ~5 A
chain of Arg290, a binding site for the adenyl group of
ATP in the pocket. From these residues onwards, domains
Ê as rigid entities towards
III and IV move by ~2±3 A
Ê ). This
domains I and II (r.m.s.d. of all atoms 2.8 A
displacement mechanism may serve for substrate recognition but also has an effect on the protomers in the two
rings of the hexamer. Although both protomers show
electron density for the APS molecule, the extent of the
conformational change is slightly different, with an
Ê for the Ca-trace.
r.m.s.d. of all atoms of 0.97 and 0.33 A
This means that they are no longer crystallographically
equivalent (Figure 2C, black arrow) and that two subunits
now constitute the asymmetric unit, which almost leads to
a doubling of the third cell axis (Table II). The crystallographic dyad axis relates two stacked hexamers. This
non-equivalence may be caused by asymmetric ligation of
ATP/APS similar to PPAT (Izard and Geerlof, 1999).
However, this possible effect of half site occupied
hexamers is very small and only indicated by the raised
B-factors of the APS molecule in one of the two subunits in
the asymmetric unit.
To obtain the enzyme±substrate analogue complex,
further X-ray investigations using 10 mM potassium
324
chlorate, which has been considered to be a competitive
inhibitor for ATPS activity (Schriever et al., 1997), were
carried out, mixed with 5 mM MgATPaS in the buffer.
The resulting electron density resembles that of the binary
complex, with the exception of new density in the active
site cleft (Figure 5C), which can clearly be interpreted as a
pyrophosphate molecule. Forming the ternary complex
E´APS´PPi, it is localized in the middle of the bowl. The
Ê from
APS and PPi phosphates are at a distance of 6.2 A
each other. We could also observe additional weak
electron density at His201-Ne, which could be interpreted
as a chlorate molecule, which could be hydrogen bonded
or even covalently bound to the nitrogen. Thus, the
inhibition could be an effect of blocking His201 and
thereby preventing the exit of the PPi molecule. The PPi is
well hydrogen bonded to APS-N6 and -N7, and to
His204-Ne. On the other side it binds to N355-Od and
-Nd, and to Ile356-O, which belongs to the conserved
ISGT motif. This module builds a partial lid on the top of
the active site bowl and may contribute to additional
pyrophosphate binding and release during the reaction
cycle. This new observation supports the suggestion of the
presence of APS in the enzyme complex.
Conserved motifs and comparison with other
nucleotidylyl transferases
As mentioned above, the active site domain II of ATPS
exhibits distinct features, like typical fold topologies and
active site design, resembling that of other nucleotidylyl
Crystal structure of ATP sulfurylase
Fig. 7. (A) Superimposition of the native (yellow) and a complex (blue) protomer showing the rigid body displacement of the domains III and IV. The
red box marks the centre of the hinge point. (B) Close-up stereoview of (A) showing the reaction centre with a nucleotide in the active site. Phe328
¯ipping by 90° causes the displacement of Pro327, Met330, Val331 and all following residues of the molecule.
transferases. Superimposition of the structure of the ATPS
binary product complex with the structure of TagD/GCT
from B.subtilis reveals strong similarities, particularly in
the active site region between H9 and S9, and also in the
327PFR motif-containing loop region after S13 (Figures
5A±C and 8A). As a result, the nucleotidylyl moiety of
APS in the ATPS complex structure possesses the same
orientation in the pocket as CTP in the active site of GCT
(Figure 8A). Although all nucleotidylyl transferases
exhibit a/b-pyrophosphorylase activity, the enzymes of
these families and superfamilies serve different biological
purposes and have, for this reason, a broad range of further
substrates for metabolic conversion with nucleoside
triphosphates. Consequently, ATPS displays neither the
P-loop GxxGxGKT/S motif of the related nucleotide and
nucleoside kinases (Saraste et al., 1990; Bork and Koonin,
1994), nor the typical PSB-loop motif TYPKSGT of the
sulfotransferase family (Kakuta et al., 1997; Bidwell et al.,
1999), which are all known to bind nucleotides like ATP or
PAPS. Additionally, ATPS exhibits no homology to the
®ngerprint motifs of the related cytidylyltransferases, like
the 113RTEGISTT, 63RYVDEVI or 8GTFDLL motifs, the
latter preceding the HWGH-box of GCT from B.subtilis
(Weber et al., 1999). Instead of this, ATPS possesses, apart
from the typical HxxH-box, its own ®ngerprint motifs,
such as the highly conserved 197RNP-, 289GRD- and
327PFR-loops, including the 356IRSGT region in the
intermediate domain III. Due to these special modules,
the architecture of the active site and the substrate
speci®city of ATPS become unique and a considerably
different nucleotide binding mode is observed.
Accordingly, not only the phosphate/sulfate binding of
APS in ATPS is highly modi®ed. For example, in ATPS as
well as in GCT, an arginine residue, which is invariant in
many nucleotidylyl transferases, is coordinated to the
respective nucleotide (Figure 8). But, while Arg113 of
the extended 113RTEGISTT-loop ful®ls this function in the
GCT structure, in the ATPS structure, the structural
counterpart Arg329, a residue of the conserved 327PFR
motif, is exposed to the solvent and is not involved in
nucleotide binding. Instead, Arg290 from the 289GRDloop binds to the nucleotidylyl moiety of APS and may
also promote transition state stabilization by binding to the
b-phosphate of ATP. Besides this, the large loop from
325
T.C.Ullrich, M.Blaesse and R.Huber
Fig. 8. (A) Stereoview of the superimposition of the active site residues of GCT (blue) and ATPS (yellow). The carbons of CTP are marked in orange.
(B) Stereoview of the active site of ATPS with sulfate and a modelled ATP molecule in the typical horseshoe conformation. Hydrogen bonds are
shown in cyan; the direction of the nucleophilic attack of the sulfate is marked in green.
Asp291 to Gly306 is not present in the GCT structure
(Figures 3B and 8A). Directing towards the active site, this
loop is also conserved in the PAPS synthetases and, due to
its positively charged character, may have a function in
channelling the reaction product APS to the APS kinase
reaction centre (Figure 4).
Catalytic residues, mechanism and
stereoselectivity
The active site residues of ATPS, Thr196, Arg197 and
Asn198 are well coordinated to the a-phosphate of APS by
hydrogen bonds (Figure 6). However, the two histidines of
the HRAH motif, which bind to the a- and b-phosphates
of a nucleotide in many nucleotidylyl transferases, are
located in the ATPS product complex far away from the
Ê
a-phosphorus of the APS, with a distance of 5.7 A
(Figures 5B and C, and 8A). This suggests that His201 and
His204 of ATPS are not directly involved in the cleavage
of the a-b-phosphodiester bond, but play a role in
pyrophosphate binding and stabilization of a productive
ATP conformation, as described for the related class I
aminoacyl-tRNA synthetases (Leatherbarrow et al., 1985;
326
Perona et al., 1993). Based on the orientation of the
adenosyl moiety in the active site of the product complex
and the sulfate position in the native structure, we
modelled an energy-minimized ATP molecule showing
the typical U-shaped (horseshoe) conformation resembling
that of numerous nucleotidylyl transferases (Figure 8B).
But in sharp contrast to the coordination of CTP in the
active site of GCT, ATP is hydrogen bonded with
its b-phosphate-O1 to His204-Nd and with -O3 to
Arg290-Nz, whereas His201-Nd coordinates to O4 of the
g-phosphate. As typical for ATP-binding enzymes, a Mg2+
ion could coordinate to the b- and g-phosphate groups
(Garcia et al., 1990) but has not been identi®ed in the
crystal structure. Due to the horseshoe-like conformation
of ATP in the pocket, this model allows an `in-line'
nucleophilic attack of the adjacent sulfate (Figure 8B). In
support of the kinetic and stereochemical investigations of
Zhang (1999), our structural data con®rm a substitution
mechanism with stereochemical inversion at the
a-phosphorus leading directly to the formation of APS,
with PPi as leaving group and without covalent adenylyl±
enzyme intermediates (Izard and Geerlof, 1999; D'Angelo
Crystal structure of ATP sulfurylase
et al., 2000). The enzyme provides suitable binding sites
for its substrates, ATP and sulfate, brings the reactive
groups in close contact and promotes the stabilization of
the nucleotide conformation most favourable for the `inline' attack of the sulfate. During the reaction cycle, it
stabilizes the pentavalent transition state of the reactants.
Unlike numerous other nucleotidylyl transferases, such as
GCT or the class I aminoacyl-tRNA synthetases, where an
active proton-donating contribution of the HxxH histidines
to the a-b-phosphate hydrolysis mechanism has been
discussed, our model con®rms that the histidines of the
HRAH motif of ATPS bind and stabilize the pyrophosphate group before and during hydrolysis of the
a-b-phosphodiester bond, and ®nally assist in the dissociation process.
Finally, the structure of the homohexameric ATPS is the
®rst structure of a member of the ATP sulfurylase family.
According to the fold of the active site domain II and its
catalytic activity, ATPS can be classi®ed as a new member
of the nucleotidylyl transferase a/b phosphodiesterase
superfamily. But, containing three independent folding
topologies in one peptide chain, this unique composition
of the protomer may suggest a new class of a/b proteins.
The structure and location of domain IV within the ATPS
protomer con®rms its evolutionary relationship to the
related APS kinase, but raises questions about its function
in ATPS. The structure of the ternary E´APS´PPi complex
rationalizes many features of the nucleotide binding and
gives clues to a deeper insight into the catalytic mechanism of ATPS. Furthermore, this work presents a starting
point for structural and functional studies on the more
complex bifunctional PAPS synthetase, where PAPS
synthesis is performed in a concerted reaction system
that synthesizes APS and channels it to the APS kinase
domain for the ®nal PAPS formation.
Materials and methods
Puri®cation, isolation and characterization
ATPS was natively puri®ed from S.cerevisiae using a modi®ed version of
a previously described protocol (Groll et al., 1997). Five hundred
grammes of cells were suspended in buffer A (20 mM Tris±HCl pH 7.5,
1 mM EDTA and 1 mM NaN3) at a 1:1 ratio to give a creamy suspension.
For cell lysis, the suspension was mixed with an equivalent volume of
glass beads (diameter 0.5 mm) and subjected to a cell disintegrator
(Biomatik, MuÈnchen) in 100 ml aliquots. After 10 min of cell breakage
(4°C, 5000 r.p.m.), the suspension was ®ltered to remove the beads and
then centrifuged (4°C, 10 000 g, 60 min). The supernatant was
ultracentrifuged at 4°C and 140 000 g for 60 min. The centrifugation
yielded a clear, deep yellow solution containing the soluble protein. After
combining the extract, ATPS was puri®ed to homogeneity in a four-step
procedure using ion exchange and af®nity chromatography as well as gel
®ltration. The extract was applied to a 300 ml Q-Sepharose column
equilibrated with buffer B (buffer A with 250 mM NaCl). The column
was washed with buffer B and proteins were eluted with a linear gradient
of 250±800 mM NaCl in a total volume of 1 l (4°C, ¯ow rate 10 ml/min,
10 ml fractions). ATPS eluted at 450 mM NaCl as monitored by 12%
SDS±PAGE. Fractions containing ATPS were pooled and loaded on a
100 ml ceramic hydroxyapatite column (Biorad, MuÈnchen) equilibrated
with 10 mM potassium phosphate pH 7.5. After washing the column with
20 mM potassium phosphate pH 7.5, ATPS eluted at 100 mM phosphate
in a 1 l gradient (10±500 mM K2HPO4/KH2PO4, 4°C, ¯ow rate 4 ml/min,
10 ml fractions). The combined fractions were dialysed against buffer C
(20 mM Tris±HCl pH 7.5, 10 mM MgCl2) and concentrated to 10±20 mg/
ml by ultra®ltration using a 100 kDa polysulfone membrane (Sartorius,
Weinheim).
The concentrated sample was loaded on a 30 ml af®nity column
(Reactive Red, Sigma), pre-equilibrated at 4°C with buffer C. The column
was washed with buffer C and elution performed with a linear 200 ml
gradient of 0±1000 mM NaCl. ATPS eluted at 500 mM sodium chloride.
The ¯ow rate was 1.5 ml/min and 10 ml fractions were collected. The
fractions were combined, dialysed against buffer A and concentrated to
20 mg/ml by ultra®ltration using 10 kDa Centripreps and Centricons
(Amicon, Hellsdorf). The protein solution was then loaded on a
Superose 6 column (Pharmacia) pre-equilibrated with buffer A. The gel
È KTA, Pharmacia) at room
®ltration was performed at an FPLC station (A
temperature with a ¯ow rate of 0.5 ml/min. ATPS was manually collected
and stored at 4°C. The size of the protomer was determined by 12%
SDS±PAGE, and that of the protein complex by native PAGE and gel
®ltration.
Crystallization was carried out at 18°C using the hanging drop vapour
diffusion method. The protein concentration was 20 mg/ml in buffer A.
After mixing protein and reservoir solution (0.5 M NaOAc, 50 mM
HEPES pH 7.5, 25 mM CdSO4) in a 2:1 ratio, crystals were obtained after
2 days. For cryo experiments, crystals were transferred from the
crystallization drop to the reservoir buffer with 20% glycerol as cryo
protectant. After 3 h of soaking, crystals were ¯ash-frozen at 100 K in a
nitrogen Oxford cryo system using a nylon loop. Heavy-atom derivative
search was performed at 18°C by dissolving heavy-atom reagents in the
cryoprotectant buffer. After 3 h of soaking, crystals could be mounted in
the cryo stream. Additional soaking experiments with ATP analogues
(5 mM MgATPgs/MgATPas) and potassium chlorate (10 mM) were
carried out under the same conditions as described above.
Crystallographic data collection
All X-ray datasets were collected at 100 K, in-house on a 345 MAR
Research Imaging Plate detector, using a Rigaku RU200 rotating-anode
Ê ), and at the wiggler beamline BW6 at
X-ray generator (l = 1.54 A
DESY/Hamburg with synchrotron radiation of different wavelengths,
using the MAR Research CCD detector (Marresearch, Hamburg). Both
systems are supplied with a nitrogen Oxford cryo system. The diffraction
data were processed with the MOSFLM program package (Leslie, 1991).
Further data reduction and truncation of structure factors were performed
with the CCP4 suite (CCP4, 1994). Statistics for all used datasets are
given in Table I.
Structure determination and re®nement
The native structure was solved by MIR using heavy-atom derivatives of
the enzyme of tantalum bromide, mercury (ethyl mercury thiosalicylate/
thiomersal) and uranyl acetate. For crystallographic data calculation, the
CCP4 program suite (CCP4, 1994) was used. Initial heavy-atom sites of
Ê were determined by difference
tantalum cluster focal point at 4 A
Patterson maps, vector veri®cation and difference Fourier maps using the
program RSPS (CCP4, 1994). Mercury and uranium sites were
determined and veri®ed by cross phasing with the localized tantalum
cluster position. Heavy-atom positions, occupancies, B-factors and initial
phases were calculated, including the anomalous diffraction data of all
three derivative datasets. The re®nement of all parameters and the ®rst
phases were calculated with MLPHARE (CCP4, 1994) and resulted in an
Ê Fourier map was
overall ®gure of merit of 0.38. The initial 2.7 A
improved by solvent ¯attening using SOLOMON (CCP4, 1994). The ®rst
secondary structure elements could be built in this map as an incomplete
poly-alanine model. All building steps were carried out with MAIN
(Turk, 1992). With this ®rst model and the derivative phases, an improved
phase combined Fourier map could be calculated, from which the initial
amino acid side chains and thus the peptide sequence could be derived. A
successive cyclic procedure of manual model building and phase
combining of MIR and model phases, combined with phase expansion
Ê , led to the ®rst complete model of the ATPS protomer. The
to 1.95 A
asymmetric unit contains one molecule of ATPS with a solvent content of
64%. This molecule was used as the starting model for the re®nement and
Ê
density modi®cation using 2Fo ± Fc and Fo ± Fc maps at 1.95 A
resolution, which were calculated with the CNS program package V1.0
(Brunger et al., 1998). The rebuilding was alternated with positional
re®nement, simulated annealing, and positional and individual B-factor
re®nement of the model. After ®nishing the re®nement of the protein
model, metal ions, ligands and water molecules were added, the latter
automatically using CNS applications and ®nal visual inspection. The
re®nement steps included the optimization of the weighing factors of the
X-ray term and led to a ®nal R-factor of the native molecule of 19.6%
(Rfree 23.1%).
The diffraction data of the enzyme±product complex with APS and
with PPi were processed in an identical manner to the apo-crystal
described above. Initial phases of the complex were obtained by
molecular replacement. Only a truncated version (residues 1±330) of
327
T.C.Ullrich, M.Blaesse and R.Huber
the re®ned coordinates of the native molecule was applied as search
model for Patterson search techniques MOLREP (CCP4, 1994). The
translation function yielded one solution with 26.9s above the mean,
clearly superior to the next peak (13.9s). From the calculated translation
function, the position of the ®rst monomer was obtained (correlation
coef®cient 22.3%, R-factor 54.4%). The rotation function yielded the
position of the second monomer in the asymmetric unit (®nal correlation
coef®cient 39.5%, ®nal R-factor 49.9%). After manual completion of the
two molecules, the initial model was improved by rigid body re®nement,
Ê resolution using
positional re®nement and simulated annealing at 2.6 A
CNS applications, and the Engh and Huber parameters (Engh and Huber,
1991). The model of the APS was built in the density using MAIN.
Manual rebuilding and further re®nement steps, including the completion
of the model with metal ions, ligands and 763 water molecules, resulted in
a ®nal R-factor of 17.6% (Rfree 22.7%). The additional electron density of
the potassium chlorate soaking experiments was ®tted with a pyrophosphate molecule. A summary of the re®nement statistics is shown in
Table II. The model with the substrate ATP was built using SYBYL
(Tripos Inc., St Louis, MO). The atomic coordinates and structure factor
amplitudes for the native ATPS structure, the binary and the ternary
product complex have been deposited in the PDB with the entry codes
1G8F, 1G8G and 1G8F.
Figure preparation
Figure 1 was prepared with ALSCRIPT (Barton, 1993) using a CGC
sequence pile-up. Figures 2, 3, 7 and 8 were prepared with MOLSCRIPT
(Kraulis, 1991) and rendered with RASTER3D (Merritt and Murphy,
1994). Figure 4 was produced with WEBLABVIEWER (www.msi.com);
Figure 5 was prepared with BOBSCRIPT (Esnouf, 1997) and rendered
with RASTER3D (Merritt and Murphy, 1994); and Figure 6 was
produced with LIGPLOT (Wallace et al., 1995).
Acknowledgements
The authors wish to thank Gleb P.Bourenkov and Hans Bartunik for
excellent support at the BW6 Beamline (Hamburg), and acknowledge
Hans-Georg Beisel for assisting the substrate modelling and John
Richardson for helpful discussions and ®nal reading of the manuscript.
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Received October 23, 2000; revised and accepted November 27, 2000
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